Single-molecule investigation of the impacts of fluorescent DNA-binding proteins on DNA mechanical properties

Abstract DNA imaging and visualization techniques are crucial in biological experiments and have also emerged as a powerful method for single-molecule studies. Traditional intercalating dyes (e.g., SYTOX, EtBr, GelRed) can stain DNA but may alter its structure and mechanical properties, and cause photocleavage. Recently, a novel fluorescent DNA-binding protein (FP-DBP) was introduced, which can stain DNA without sequence preference and without inducing photocleavage. In this study, using a custom-built magnetic tweezers system, we performed DNA stretching, twisting and unzipping experiments to compare the mechanical properties of DNA with and without two kinds of intercalating dyes (SYTOX Orange and GelRed) and mCherry FP-DBP. Our results show that mCherry FP-DBP does not affect DNA structure or mechanics, unlike SYTOX Orange and GelRed, making FP-DBP a promising tool for DNA visualization in single-molecule experiments.